Antibacterial agents

ABSTRACT

The compounds of the formula: ##STR1## and salts and esters thereof are useful agents for the treatment of bacterial infections either alone or in combination with a penicillin or cephalosporin derivative. The preceding compounds may be prepared by the hydrogenation of a compound of the formula: ##STR2## wherein R is a hydrogen atom or an acyl group or a salt or ester thereof.

CROSS-REFERENCE

This is a division of Ser. No. 857,112 filed Dec. 5, 1977 which itselfis a divisional of Ser. No. 669,697, filed Mar. 23, 1976.

The present invention relates to novel β-lactam containing compoundsuseful in anti-bacterial therapy, to compositions containing these novelcompounds and to the method of their preparation.

Belgian Pat. No. 827926 discloses inter alia that the compound of theformula (I): ##STR3## and its salts and esters possess anti-bacterialand β-lactamase inhibitory activity. The compound of the formula (I) isdesignated clavulanic acid. Acylated derivatives of the above compoundsare disclosed in Netherlands Patent Application No. 75/12348 and WestGerman Patent Application No. 2555626 discloses inter alia isoclavulanicacid and its salts and esters which compound isoclavulanic acid has theformula (II): ##STR4##

Isoclavulanic acid and its salts and esters also have antibacterial andβ-lactamase inhibitory activity. A further group of compounds withuseful anti-bacterial and β-lactamase inhibiting properties has now beendiscovered.

Accordingly the present invention provides the compounds of the formula(III): ##STR5## and salts and esters thereof.

The stereochemistry at C-2 and C-5 of the compounds of formula (III) isthe same as that found in naturally occurring penicillins.

The two isomeric acids of the formulae (IV) and (V): ##STR6## aretherapeutic agents and are useful intermediates in the formation oftheir esters but in general their pharmaceutically acceptable salts aremore favoured because of their improved stability. The compound of theformula (IV) is designated herein as deoxyclavulanic acid and thecompound of the formula (V) is designated herein isodeoxyclavulanicacid.

In general deoxyclavulanic acid and its derivatives form a morefavourable aspect of this invention than does isodeoxyclavulanic acidand its derivatives because of their generally more facile production.

Suitable salts of the compounds of the formula (III) includeconventional pharmaceutically acceptable salts such as the sodium,potassium, calcium, magnesium, ammonium and conventional substitutedammonium salts formed with benzylpenicillin such as the 1-ephenamine,procaine, benzathine and the like salts.

Particularly suitable salts of deoxyclavulanic acid andisodeoxyclavulanic acid include their sodium and potassium salts.

Preferred salts of this invention include those of the formulae (VI) and(VII): ##STR7## The sodium salt of deoxyclavulanic acid is aparticularly suitable compound of this invention.

Non-pharmaceutically acceptable salts of the compounds of the formula(III) can also be useful as they can serve as intermediates in thepreparation of esters of the compounds of formula (III); for example, byreaction with pivaloyloxymethyl chloride to give a useful antibacterialagent.

Suitable esters of the compounds of formula (III) include those of theformula (VIII): ##STR8## wherein R is an organic group such that thealcohol ROH is pharmaceutically acceptable.

It is envisaged that the esters of deoxyclavulanic acid anddeoxyisoclavulanic acid owe much of their antibacterial activity totheir ability to act as pro-drugs for deoxyclavulanic andisodeoxyclavulanic acids and their salts. Thus preferred esters arethose which are convertible to the corresponding acid or its salts underphysiological conditions.

Particularly suitable esters of the compounds of the formula (VIII)include those of the formula (IX): ##STR9## wherein R is as defined inrelation to formula (VIII).

Suitable groups R for inclusion in the compounds of formula (VIII) and(IX) include alkyl, alkenyl, alkynyl, aryl, arylalkyl or other similargroups any of which may be substituted if desired.

In order not to increase the molecular weight to an unreasonable extent,groups R do not normally include more than 16 carbon atoms, moresuitably not more than 12 carbon atoms and most suitably, not more than8 carbon atoms. Generally the CO₂ R group is such that the compound ofthe formula (VIII) has a molecular weight of not more than 400.

Preferably, the group R is notionally derived from an alcohol ROH whichis pharmaceutically acceptable. Suitable groups R include methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl,methylcyclopentyl, methylcyclohexyl, cyclopentylmethyl,cyclohexylmethyl, benzyl, benzhydryl, phenylethyl, naphthylmethyl,naphthyl, phenyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl,2,2,2-trifluoroethyl, acetylmethyl, benzoylmethyl, 2-methoxyethyl,p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,m-chlorobenzyl, 6-methoxynaphthyl-2-methyl, p-chlorophenyl,p-methoxyphenyl, β-2'-pyridylethyl or like group.

Suitable readily in-vivo hydrolysable ester groups CO₂ R include but arenot limited to acyloxyalkyl and lactone groups such as those representedby the sub-formulae (a) and (b): ##STR10## wherein A₁ is a hydrogen atomor a methyl group; A₂ is a hydrogen atom or a methyl, ethyl or phenylgroup; A₃ is an alkyl or alkoxyl group of 1-6 carbon atoms or a phenylor benzyl group; A₄ is --CH₂ CH₂ --, --CH:CH--, ##STR11## and X is anoxygen or sulphur atom. Most suitably X is an oxygen atom and A₂ is amethyl or t-butyl group and A₄ is a phenylene group.

A further particularly suitable sub-group of esters of formulae (VIII)or (IX) are those wherein R is a group R¹ or CHR² R³ wherein R¹ is ahydrocarbon group of 1-9 carbon atoms optionally substituted by halogen,lower alkoxy, lower acyl, hydroxy or lower acyloxy groups and R² is anoptionally substituted phenyl group and R³ is an optionally substitutedphenyl group.

The term `lower` used herein means the group contains up to 6 carbonatoms. The term `optionally substituted phenyl` includes a phenyl groupand a phenyl group substituted by a halogen atom or a lower alkyl orlower alkoxy group.

An alternative aspect of the present invention provides a pharmaceuticalcomposition which contains a compound of the formula (III) or apharmaceutically acceptable salt or ester thereof and a pharmaceuticallyacceptable carrier. Favourably such compositions contain apharmaceutically acceptable salt or in-vivo hydrolysable ester of acompound of the formula (III).

More suitably, the pharmaceutical composition of this invention willcontain a pharmaceutically acceptable salt of the compound of theformula (III). The compositions of this invention will normally beadapted for administration to humans and other mammals, for example, inconventional modes of treatment of diseases of the urinary tract,respiratory system and soft tissues as well as diseases such as otitismedia in humans and mastitis in domestic animals and the like.

Suitable forms of the compositions of this invention include tablets,capsules, creams, syrups, suspensions, solutions, reconstitutablepowders and sterile forms suitable for injection or infusion may beused. Such compositions may contain conventional pharmaceuticallyacceptable materials such as diluents, binders, colours, flavours,preservatives, disintegrants and the like in accordance withconventional pharmaceutical practice and the arts of formulatingantibiotic compositions.

Compositions adapted for oral administration may also comprise abuffering agent or may be protected from gastric juice in otherconventional manner if so desired.

The compound of formula (III) may be present in the composition as soletherapeutic agent or it may be present together with other therapeuticagents such as a β-lactam antibiotic. Suitable β-lactam antibiotics forinclusion in such compositions include not only those known to besusceptible to β-lactamases but also those which have a degree ofintrinsic resistance to β-lactamases. Thus, suitable β-lactamantibiotics for inclusion in the composition of this invention includebenzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin,propicillin, hetacillin, ampicillin, amoxycillin, ticarcillin,cephaloridine, cephalothin, cephalexin, cephaloglycin, cephamandole andin-vivo hydrolysable esters of such compounds such as the phenyl, tolyland 5-indanyl esters of carbenicillin and ticarcillin, the acetoxymethylester of benzylpenicillin and the acetoxymethyl, pivaloyloxymethyl andphthalidyl esters of ampicillin, amoxycillin, cephaloglycin, cephalexin,mecillinam and the like or salts of such compounds.

When present in a pharmaceutical composition together with a β-lactamantibiotic, the ratio of the compound of formula (III) or its salt orester present to β-lactam antibiotic present may be from, for example,20:1 to 1:5, such as 10:1 to 1:3, and advantageously may be 5:1 to 1:2,for example, 3:1 to 1:1.

The total quantity of antibacterial agents present in any unit dosageform will normally be between 50 and 1500 mg and will usually be between100 and 1000 mg. However, injectable or infusable compositions maycontain greater quantities if desired, for example, 4 g or more ofactive material. Normally, between 50 and 6000 mg of the compositions ofthis invention will be administered each day of treatment but moreusually between 500 and 3000 mg of the composition of the invention willbe administered per day. In general the equivalent of not more than 2000mg of a compound of the formula (III) will be administered per day, forexample, 100-1000 mg.

In a further aspect this invention provides synergistic compositionswhich contain a compound of the formula (III) or a pharmaceuticallyacceptable salt or ester thereof and ampicillin, amoxycillin or apro-drug for ampicillin or amoxycillin. Such compositions are preferablyadapted for administration to humans and contain 50-500 mg of a salt orin-vivo hydrolysable ester of a compound of the formula (III) and200-1000 mg of the penicillin. Particularly suitable forms of thepenicillins for inclusion in orally administrable forms of suchcompositions include ampicillin trihydrate, amoxycillin trihydrate,acetylamoxycillin trihydrate, anhydrous ampicillin, ampicillinpivaloyloxymethyl ester and ampicillin phthalidyl ester or salts such asthe hydrochloride of such esters. Particularly suitable forms of thepenicillins for inclusion in injectable forms include sodium ampicillinand sodium amoxycillin, sodium ampicillin being preferred. Suchcompositions may be used in treating infections of the urinary tract andrespiratory tract and are particularly useful in treating infections dueto strains of Klebsiella aeroginosa, Proteus, or E. coli.

In a further aspect this invention provides synergistic compositionswhich contain a compound of the formula (III) or a pharmaceuticallyacceptable salt or ester thereof and carbenicillin or ticarcillin ortheir salts or a pro-drug for carbenicillin or ticarcillin such ascarbenicillin phenylα-ester, carbenicillin 5-indanylα-ester, ticarcillinphenylα-ester or ticarcillin tolylα-ester or their salts. Suchcompositions are preferably adapted for administration to humans andcontain 50-1500 mg of a salt or in-vivo hydrolysable ester of a compoundof the formula (III) and 200-1500 mg of the penicillin. Suchcompositions may be used in treating infections of the urinary tract.

The preceding compositions preferably contain a pharmaceuticallyacceptable salt of a compound of the formula (III) such as the sodium orpotassium salt, for example, a compound of the formula (VI).

In a further aspect this invention provides a process for thepreparation of a compound of the formula (III) as hereinbefore definedor a salt or ester thereof which process comprises the hydrogenation ofa corresponding compound of the formula (X): ##STR12## or a salt orester thereof wherein R₆ is a hydrogen atom or an acyl group.

It is frequently particularly convenient to use a compound of theformula (X) wherein R₆ is a hydrogen atom. It is also frequentlyconvenient to use a compound of the formula (X) in the form of a saltthereof.

Normally, such a reaction takes place in the presence of a transitionmetal containing catalyst such as palladium, platinum oxide or the like.A particularly suitable catalyst is palladium on charcoal, for example,10% palladium on charcoal.

The catalyst employed is suitably in highly active form, for example,the sort obtained by using a fresh batch of catalyst.

Most suitably the weight of catalyst (as total 10% palladium andcharcoal or the equivalent) present is at least 1/3 of the weight of thecompound of formula (X) or salt or ester present. It is advantageous tohave at least as much catalyst present as compound of formula (X)present, especially for those compounds wherein R₆ is H.

The process of this invention normally takes place at a non-extremetemperature; for example, the reaction may take place in a lower alkanolat a temperature of -10° C. to +50° C., more usually from 0° C. to 25°C., for example, from 5° C. to 20° C.

The process of this invention normally takes place in an inert solventsuch as a lower alkanol, water or an aqueous alkanol. Most suitably thesolvent employed is a lower alkanol such as methanol or ethanol. Forthose compounds of the formula (X) wherein R₆ is H water miscible etherssuch as tetrahydrofuran are also suitable solvents but such ethersolvents are not generally suitable for use when R₆ is an acyl group.

An elevated, medium or low pressure of hydrogen may be used in thisreaction. Generally, it is preferred to use an atmospheric or slightlysuper atmospheric pressure of hydrogen.

A preferred form of the process of this invention comprises thehydrogenation of clavulanic acid or a salt or hydrogenisable esterthereof in the presence of a palladium catalyst. Such a process leads tothe preparation of a compound of the formula (IV) or a salt thereof. Ifa salt is required and the clavulanic acid is not already in salt form abase such as sodium bicarbonate or the like may be included in thereaction medium.

Hydrogenation of isoclavulanic acid or a derivative thereof frequentlyleads to a deoxyisoclavulanic acid derivative contaminated with acorresponding deoxyclavulanic acid derivative. A purer product may thenbe obtained by chromatography.

The nature of the acyl group R₆ which may be present in a compound ofthe formula (X) is relatively unimportant as long as it does not lead tothe rapid breakdown of the compound of formula (X). Suitable acylderivatives are described in Netherlands Application No. 75/12348.Particularly suitable acyl groups R₆ contain up to 16 carbon atoms andmay be optionally substituted by groups such as halogen, lower alkoxy,lower alkoxycarbonyl, lower acyloxy, hydroxy and the like. Most suitablysuch acyl groups are unsubstituted or substituted by non-reactive groupsonly.

Esters of the compounds of formula (III) may be prepared by the reactionof a compound of the formula (III) or a salt thereof with an alcohol ROHor a compound of the formula RQ where Q is a good leaving group such asa chlorine, bromine or iodine atom or an activated ester group or asulphonic ester such as a mesylate or tosylate group or otherconventional good leaving group. Alternatively, the acid of the formula(III) may be treated with a diazocompound such as diazomethane or thelike or with an alcohol ROH in the presence of a dehydrating agent suchas a carbodiimide or its chemical equivalent.

The reaction with RQ¹ is normally carried out in an organic solvent ofrelatively high dielectric constant such as dimethylformamide, acetone,dioxane, tetrahydrofuran or the like and at a non-extreme temperaturesuch as -5° C. to 100° C., more usually +5° C. to 30° C., for example,at ambient temperature.

The reaction of an acid of formula (III) with a diazoalkane is a mildmethod of making alkyl, aralkyl or similar esters. The diazotizationreaction may be performed under conventional reaction conditions, forexample at a non-extreme temperature and in a conventional solvent. Suchreactions are normally carried out at between -5° C. and 100° C., moreusually from 5° C. to 30° C., for example at ambient temperature.Suitable solvents for this reaction include lower alkanols such asmethanol and ethanol and solvents such as tetrahydrofuran, dioxane andthe like. Ethanol has proved a particularly useful solvent for thisreaction.

The reaction of an acid of formula (III) with an alcohol in the presenceof a condensation promoting agent will normally take place in an inertorganic solvent such as dichloromethane or acetonitrile. This reactionis usually carried out at an ambient or depressed temperature, forexample at -10° C. to +22° C., more usually -5° C. to +18° C., forexample initially at 0° C. and thereafter gradually warming to about 15°C. The condensation promoting agent used is normally one which removeswater from the reaction mixture. Suitable agents include carbodiimides,carbodiimidazoles or equivalent reagents. Dicyclohexylcarbodiimide hasproved to be a particularly suitable condensation promoting agent foruse in this process.

Other less suitable methods of ester formation include (a) removal ofthe elements of carbon dioxide from a compound of the formula (XI):##STR13## wherein R₇ is an inert organic group; and also (b) reaction ofa compound of the formula (XI) with alcohol ROH.

The compound of the formula (XI) may be prepared by the reaction of asalt of a compound of the formula (III) with Cl.CO.O.R₇ or the chemicalequivalent thereof.

Salts of the compounds of the formula (III) may be prepared by thehydrolysis of an ester of a compound of the formula (III). Generallythis may be brought about by keeping the ester of the compound offormula (III) in an aqueous medium maintained at pH of about 7-9 for upto one hour. Certain reactive esters such as the pivaloyloxymethyl,acetoxymethyl, phthalidyl and like esters hydrolyse in a few minuteswhen maintained in an aqueous medium at a pH of about 6-8.

The following Examples illustrate the invention:

EXAMPLE 1 Sodium Deoxyclavulanate ##STR14##

Benzyl clavulanate (220 mg) in ethanol (20 ml) was hydrogenated over 10%Pd/C (70 mg) and sodium hydrogen carbonate (60 mg) for 60 minutes. Thecatalyst was filtered, washed with water and then ethanol and thecombined filtrates were evaporated. This material was chromatographed ona silica gel column with n-butanol/ethanol/water; 4:1:13/4 and thefastest moving component was collected. The solvents were removed underlow pressures to yield the sodium salt of deoxyclavulanic acid.

I.r. (KBr): 1780, 1700, 1605 cm⁻¹ ; n.m.r. (D₂ O): 1.52 (3H, dd, J 7 Hz,J' 1.5 Hz); 2.98 (1H, d, J 18 Hz, 6β-CH); 3.52 (1H, dd, J 18 Hz, J' 2.5Hz, 6α-CH); 4.5-4.9 (m, obscured by HOD peak); 5.64 (1H, d, J 2.5 Hz,5-CH).

(The sodium salt of clavulanic acid was also obtained from the column onfurther elution).

EXAMPLE 2 Sodium Deoxyclavulanate ##STR15##

Benzyl clavulanate (8.25 g) was dissolved in tetrahydrofuran (75 ml). Tothe solution was added 10% palladium on charcoal (8.25 g) and themixture was hydrogenolysed at room temperature with vigorous shaking andusing 1 atmosphere pressure of hydrogen for 30 minutes. The suspensionwas filtered and the filtrate was treated with a solution of sodiumbicarbonate (2.39 g) dissolved in the minimum amount of water. Thesolution was concentrated under reduced pressure on a rotary evaporatorat room temperature and the residue was triturated with acetone andether to give a pale yellow solid (4.7 g).

EXAMPLE 3 Sodium Deoxyclavulanate ##STR16##

Benzyl phenoxyacetylclavulanate (140 mg) was dissolved in ethanol/ethylacetate (5:1, 6 ml) and sodium bicarbonate (56 mg) and 10% palladium oncharocal (47 mg) were added to the solution. The solution washydrogenated at ambient temperature (˜18° C.) for 15 minutes. Thecatalyst was filtered off and washed well with water. The filtrate andwashings were combined and evaporated to dryness to give a quantitativeyield of sodium deoxyclavulanate. Sodium deoxyclavulanate may beseparated from the mixture with sodium phenoxyacetate by careful columnchromatography using silica gel and eluting with butanol/ethanol/water.(Physical characteristics of product as in Example 1).

The preceding example may be varied by replacing the benzylphenoxyacetylclavulanate with equivalent amounts of benzylacetylclavulanate, benzyl α-phenyloxycarbonylphenylacetylclavulanate,p-bromobenzyl phenoxyacetylclavulanate and the like. The precedingexample may also be varied by replacing the benzylphenoxyacetylclavulanate with an equivalent amount of benzylα-benzyloxycarbonylphenylacetamidoclavulanate and increasing the amountof sodium bicarbonate to 2 equivalents.

EXAMPLE 4 p-Bromobenzyl Deoxyclavulanate ##STR17##

A solution of p-bromobenzyl bromide (50 mg) was added to a solution ofsodium deoxyclavulanate (10 mg) in dimethylformamide (0.5 ml) and themixture was kept at ambient temperature (about 18° C.) for 2 hours. Thereaction mixture was fractionated on silica gel eluting with ethylacetate/hexane (1:4) to yield p-bromobenzyl deoxyclavulanate (as an oil)on evaporation.

I.r. (CHCl₃): 1790, 1740, 1695 cm⁻¹ ; N.m.r. (CDCl₃): 1.62 (3H, dd, J 7Hz, J' 1.4 Hz, CH₃); 2.95 (1H, dd, J 17 Hz, J' 1.0 Hz, 6β-CH); 3.48 (1H,dd, J 17 Hz, J' 2.6 Hz, 6α-CH); 4.58 (1H, dq, J 7 Hz, J' 1 Hz, ═CHCH₃);5.03 (1H, dd, J 1.4 Hz, J' 1.0 Hz, 3-CH); 5.12 (2H, s, CO₂ CH₂ --); 5.65(1H, dd, J 2.6 Hz, J' 1.0 Hz, 5-CH); 3.38 (4H, ABq, J 8.5 Hz, aromaticprotons).

The preceding example may be repeated replacing p-bromobenzyl bromidewith an equivalent quantity of methyl iodide, ethyl bromide,1-bromo-2-methoxyethane, pivaloyloxymethyl chloride, phthalidyl bromide,1-chloro-2-thiomethylethane, 1-chloro-2-phenylsulphonylethane,1-bromononane, 4-methoxybenzylbromide, benzylbromide, benzylchloride,phenacetyl bromide or the like.

EXAMPLE 5 Sodium Deoxyisoclavulanate ##STR18##

Benzyl isoclavulanate (50 mg) in tetrahydrofuran (0.5 ml) washydrogenated at room temperature (˜18° C.) and atmospheric pressureusing 10% palladium on charcoal (50 mg) as catalyst. After 30 minutesthe catalyst was filtered off and an equivalent amount of aqueous sodiumbicarbonate added. The solvent was removed by evaporation and theresidue triturated with ethanol, acetone and acetone/ether to give theproduct as an off-white solid (20 mg). (The n.m.r. spectrum in D₂ Oshowed that the title compound was contaminated with sodiumdeoxyclavulanate).

EXAMPLE 6 Pharmacology

Sodium deoxyclavulanate did not appear to produce any overt toxiceffects in mice when administered intra peritoneally at 500 mg/kg.

The antibacterial and synergistic properties of sodium deoxyclavulanateare illustrated by the following in-vitro results:

    ______________________________________                                                          Minimum Inhibitory Concen-                                                    tration of Sodium Deoxycla-                                 Organism          vulanate (μg/ml)                                         ______________________________________                                        Bacillus subtilis A                                                                             62.5                                                        Enterobacter cloacae N1                                                                         125                                                         Escherichia coli 10418                                                                          62.5                                                        Klebsiella aerogenes A                                                                          62.5                                                        Proteus mirabilis C 977                                                                         125                                                         Pseudomonas aeruginosa A                                                                        1000                                                        Salmonella typhimurium CT10                                                                     125                                                         Serratia marcescens US 39                                                                       125                                                         Staph. aureus Oxford                                                                            15.6                                                        Staph. aureus Russell                                                                           31                                                          ______________________________________                                    

    ______________________________________                                                       Minimum Inhibitory Concentra-                                                 tion (μg/ml) of Ampicillin in                                              Presence of Various Concentra-                                                tions of Sodium Deoxyclavul-                                                  anate (μg/ml)                                               Organism         0       1     5     20                                       ______________________________________                                        Staphylococcus aureus Russell                                                                   500    0.8   0.08  *                                        Klebsiella aerogenes E70                                                                       >500    0.8   0.2-0.4                                                                             0.1-0.02                                 ______________________________________                                         *Inhibition produced by sodium deoxyclavulanate alone at this                 concentration                                                            

What is claimed is:
 1. A pharmaceutical composition for treatingbacterial infections in humans and animals which comprises asynergistically effective amount of a compound of the formula (III):##STR19## or a pharmaceutically acceptable salt thereof, and anantibacterially effective amount of benzylpenicillin or theacetoxymethyl ester thereof, in combination with a pharmaceuticallyacceptable carrier, the weight ratio of said compound orpharmaceutically acceptable salt thereof to benzylpenicillin or saidester thereof being 20:1 to 1:5.
 2. A composition according to claim 1wherein the compound is in the form of a pharmaceutically acceptablesalt wherein said salt is selected from the group consisting of thesodium, potassium, calcium, magnesium, ammonium, ephenamine, procaineand benzathine salts.
 3. A composition according to claim 1 wherein thepharmaceutically acceptable salt is of the formula (IV) or (V):##STR20##
 4. A composition according to claim 3 wherein the salt is thesodium or potassium salt of compound (IV) or (V).
 5. A compositionaccording to claim 1 wherein the compound is in the form of a salt ofthe formula (VI) or (VII): ##STR21##
 6. A composition according to claim5 wherein the compound is in the form of the sodium salt of the compound(VI).
 7. A composition according to claim 1 wherein the ratio is 10:1 to1:3.
 8. A composition according to claim 1 wherein the ratio 5:1 to 1:2.9. A composition according to claim 1 wherein the ratio 3:1 to 1:1. 10.A method of treating bacterial infection in humans and animals whichcomprises administering to humans or animals in need thereofsynergistically effective amount of a compound of the formula (III):##STR22## or a pharmaceutically acceptable salt thereof, and anantibacterially effective amount of benzylpenicillin or theacetoxymethyl ester thereof, the weight ratio of said compound orpharmaceutically acceptable salt thereof and said benzylpenicillin orester thereof being 20:1 to 1:5.
 11. A method according to claim 10wherein the compound is a pharmaceutically acceptable salt of theformula (IV) or (V): ##STR23##
 12. A method according to claim 11wherein the compound is a sodium or potassium salt.
 13. A methodaccording to claim 10 wherein the compound is a salt of the formula (VI)or (VII): ##STR24##
 14. A method according to claim 13 wherein thecompound is a sodium salt of the formula (VI).
 15. A method according toclaim 10 wherein the ratio is 10:1 to 1:3.
 16. A method according toclaim 10 wherein the ratio 5:1 to 1:2.
 17. A method according to claim10 wherein the ratio 3:1 to 1:1.